U.S. patent application number 10/402333 was filed with the patent office on 2003-12-18 for nucleic acid array preparation using purified phosphoramidites.
This patent application is currently assigned to Affymetrix, Inc.. Invention is credited to Barone, Dale, Bury, Paul, Carroll, Robert, McGall, Glenn, Trulson, Mark.
Application Number | 20030232361 10/402333 |
Document ID | / |
Family ID | 29741225 |
Filed Date | 2003-12-18 |
United States Patent
Application |
20030232361 |
Kind Code |
A1 |
Barone, Dale ; et
al. |
December 18, 2003 |
Nucleic acid array preparation using purified phosphoramidites
Abstract
Improved nucleic acid arrays are provided which have been
prepared using nucleoside phosphoramidites having reduced levels of
interfering phosphoramidite impurities.
Inventors: |
Barone, Dale; (San Jose,
CA) ; McGall, Glenn; (Mountain View, CA) ;
Trulson, Mark; (San Jose, CA) ; Bury, Paul;
(San Bruno, CA) ; Carroll, Robert; (Mountain View,
CA) |
Correspondence
Address: |
HAMILTON, BROOK, SMITH & REYNOLDS, P.C.
530 VIRGINIA ROAD
P.O. BOX 9133
CONCORD
MA
01742
US
|
Assignee: |
Affymetrix, Inc.
Santa Clara
CA
|
Family ID: |
29741225 |
Appl. No.: |
10/402333 |
Filed: |
March 27, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10402333 |
Mar 27, 2003 |
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09810419 |
Mar 15, 2001 |
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10402333 |
Mar 27, 2003 |
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09608691 |
Jun 29, 2000 |
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09608691 |
Jun 29, 2000 |
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08630427 |
Apr 3, 1996 |
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6156501 |
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08630427 |
Apr 3, 1996 |
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08440742 |
May 10, 1995 |
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08440742 |
May 10, 1995 |
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PCT/US94/12305 |
Oct 26, 1994 |
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PCT/US94/12305 |
Oct 26, 1994 |
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08284064 |
Aug 2, 1994 |
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08284064 |
Aug 2, 1994 |
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08143312 |
Oct 26, 1993 |
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60190166 |
Mar 17, 2000 |
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Current U.S.
Class: |
506/16 ;
427/2.11; 435/6.11; 506/30 |
Current CPC
Class: |
B01J 2219/00612
20130101; B01J 2219/00529 20130101; C40B 40/06 20130101; B01J
2219/00722 20130101; C07B 2200/11 20130101; B01J 2219/00432
20130101; B01J 2219/00639 20130101; C07H 21/00 20130101; B01J
2219/00711 20130101; B01J 2219/00637 20130101; B01J 2219/00617
20130101; B01J 2219/00621 20130101; B82Y 30/00 20130101; B01J
2219/00608 20130101; B01J 19/0046 20130101; C40B 60/14 20130101;
B01J 2219/0061 20130101; B01J 2219/00626 20130101; B01J 2219/00659
20130101 |
Class at
Publication: |
435/6 ;
427/2.11 |
International
Class: |
C12Q 001/68; B05D
003/00 |
Claims
What is claimed is:
1. A method of preparing a nucleic acid array on a support
comprising preparing a nucleic acid array on a support, wherein
each nucleic acid occupies a separate known region of the support,
said method comprising contacting said support with protected
nucleoside phosphoramidite monomers having less than about 1 mole %
of a phosphoramidite contaminant, as measured by .sup.1H NMR
spectrometry, selected from the group consisting of
(MeO)(NCCH.sub.2CH.sub.2O)PN(iPr).sub.2,
(MeO)P(N(iPr).sub.2).sub.2, (MeO).sub.2PN(iPr).sub.2, and
(NCCH.sub.2CH.sub.2O).sub.2PN(iPr).sub.2.
2. A method in accordance with claim 1, said method further
comprising: (a) activating a region of the support; (b) attaching a
nucleotide to said region, said nucleotide having a masked reactive
site linked to a protecting group; (c) repeating steps (a) and (b)
on other regions of said support whereby each of said other regions
has bound thereto another nucleotide comprising a masked reactive
site linked to a protecting group, wherein said another nucleotide
may be the same or different from that attached in step (b); (d)
removing the protecting group from one of the nucleotides bound to
one of the regions of the support to provide a region bearing a
nucleotide having an unmasked reactive site; (e) binding an
additional nucleotide to the nucleotide having an unmasked reactive
site; and (f) repeating steps (d) and (e) on regions of the support
until a desired plurality of nucleic acids is synthesized, each
nucleic acid occupying separate known regions of the support;
wherein said phosphoramidite contaminant is present in an amount of
less than about 0.5 mole % as measured by .sup.1H NMR
spectrometry.
3. A method in accordance with claim 1, wherein said method
comprises the sequential steps of: (a) generating a pattern of
light and dark areas by selectively irradiating at least a first
area of a surface of a substrate, said surface comprising
immobilized nucleotides on said surface, said nucleotides capped
with a photoremovable protecting group, without irradiating at
least a second area of said surface, to remove said protecting
group from said nucleotides in said first area; (b) simultaneously
contacting said first area and said second area of said surface
with a first nucleotide to couple said first nucleotide to said
immobilized nucleotides in said first area, and not in said second
area, said first nucleotide capped with said photoremovable
protecting group; (c) generating another pattern of light and dark
areas by selectively irradiating with light at least a part of said
first area of said surface and at least a part of said second area
to remove said protecting group in said at least a part of said
first area and said at least a part of said second area; (d)
simultaneously contacting said first area and said second area of
said surface with a second nucleotide to couple said second
nucleotide to said immobilized nucleotides in at least a part of
said first area and at least a part of said second area; and (e)
performing additional irradiating and nucleotide contacting and
coupling steps so that a matrix array of at least 100 nucleic acids
having different sequences is formed on said support.
4. A method in accordance with claim 1, wherein said contaminant is
present in an amount of less than about 0.2 mole % as measured by
.sup.1H NMR spectrometry.
5. A method in accordance with claim 1, wherein said protected
nucleoside phosphoramidite monomers have the formula: 3wherein B is
a member selected from the group consisting of adenine, guanine,
thymine, cytosine, uracil and analogs thereof, R is a member
selected from the group consisting of hydrogen, hydroxy, protected
hydroxy, halogen and alkoxy; P is a phosphoramidite group; and PG
is a photoremovable protecting group.
6. A method in accordance with claim 5, wherein B is selected from
the group consisting of adenine, guanine, cytosine and thymine and
R is hydrogen.
7. A method in accordance with claim 5, wherein said array
comprises at least 10 nucleic acids having a different
sequence.
8. A method in accordance with claim 5, wherein said array
comprises at least 100 nucleic acids having a different
sequence.
9. A method in accordance with claim 5, wherein said array
comprises at least 1000 nucleic acids having a different
sequence.
10. A method in accordance with claim 5, wherein said array
comprises at least 10,000 nucleic acids having a different
sequence.
11. A method in accordance with claim 5, wherein said array
comprises at least 100,000 nucleic acid having a different
sequence.
12. A method in accordance with claim 3, wherein each nucleic acid
having a different sequence is in a region having an area of less
than about 1 cm.sup.2.
13. A method in accordance with claim 3, wherein each nucleic acid
having a different sequence is in a region having an area of less
than about 1 mm.sup.2.
14. A method in accordance with claim 5, wherein said
phosphoramidite contaminant is present in an amount of less than
0.2 mole % as measured by .sup.1H NMR spectrometry.
15. A method in accordance with claim 5, wherein B is selected from
the group consisting of adenine, guanine, cytosine and thymine, R
is hydrogen, and said phosphoramidite contaminant is present in an
amount of less than 0.2 mole % as measured by .sup.1H NMR
spectrometry.
16. A method in accordance with claim 5, wherein B is selected from
the group consisting of adenine, guanine, cytosine and thymine, R
is hydrogen, PG is MeNPOC and said phosphoramidite contaminant is
present in an amount of less than 0.2 mole % as measured by .sup.1H
NMR spectrometry.
17. A method in accordance with claim 5, wherein B is selected from
the group consisting of adenine, guanine, cytosine and thymine, R
is hydrogen, PG is MeNPOC, P is
--P(OCH.sub.2CH.sub.2CN)N(iPr).sub.2 and said phosphoramidite
contaminant is present in an amount of less than 0.2 mole % as
measured by .sup.1H NMR spectrometry.
18. A nucleic acid array prepared by the method of claim 1.
19. A nucleic acid array prepared by the method of claim 5.
20. A nucleic acid array prepared by the method of claim 17.
Description
RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S.
application Ser. No. 09/810,419, filed Mar. 15, 2001, which claims
the benefit of U.S. application Ser. No. 60/190,166, filed Mar. 17,
2000, each of which is incorporated herein by reference. This
application is also a continuation-in-part of U.S. application Ser.
No. 09/608,691, filed Jun. 29, 2000, which is a continuation of
U.S. application Ser. No. 08/630,427, filed Apr. 3, 1996, now U.S.
Pat. No. 6,156,501, which is a continuation-in-part of U.S.
application Ser. No. 08/440,742, filed May 10, 1995, now abandoned,
which is a continuation-in-part of International Application No.
PCT/US94/12305, filed Oct. 26, 1994, published in English and
designating the U.S., which is a continuation-in-part of U.S.
application Ser. No. 08/284,064, filed Aug. 2, 1994, now abandoned,
which is a continuation-in-part of U.S. application Ser. No.
08/143,312, filed Oct. 26, 1993, now abandoned, each of which is
incorporated by reference.
BACKGROUND OF THE INVENTION
[0002] The present invention relates to improved methods for
preparing support bound nucleic acid arrays. More particularly, the
invention relates to methods of preparing the arrays wherein
impurities that can affect the variability and performance of the
arrays are excluded from reagent solutions.
[0003] Substrate-bound nucleic acid arrays, such as the Affymetrix
DNA Chip, enable one to test hybridization of a target nucleic acid
molecule to many thousands of differently sequenced nucleic acid
probes at feature densities greater than about five hundred per 1
cm.sup.2. Because hybridization between two nucleic acids is a
function of their sequences, analysis of the pattern of
hybridization provides information about the sequence-of the target
molecule. The technology is useful for de novo sequencing and
resequencing of nucleic acid molecules and also has important
diagnostic uses in discriminating genetic variants that may differ
in sequence by one or a few nucleotides. For example,
substrate-bound nucleic acid arrays are useful for identifying
genetic variants of infectious diseases, such as HIV, or genetic
diseases, such as cystic fibrosis.
[0004] In one version of the substrate-bound nucleic acid array,
the target nucleic acid is labeled with a detectable marker, such
as a fluorescent molecule. Hybridization between a target and a
probe is determined by detecting the fluorescent signal at the
various locations on the substrate. The amount of signal is a
function of the thermal stability of the hybrids. The thermal
stability is, in turn, a function of the sequences of the
target-probe pair: AT-rich regions of DNA melt at lower
temperatures than GC-rich regions of DNA. This differential in
thermal stabilities is the primary determinant of the breadth of
DNA melting transitions, even for nucleic acids.
[0005] Depending upon the length of the nucleic acid probes, the
number of different probes on a substrate, the length of the target
nucleic acid, and the degree of hybridization between sequences
containing mismatches, among other things, a hybridization assay
carried out on a substrate-bound nucleic acid array can generate
thousands of data points of different signal strengths that reflect
the sequences of the probes to which the target nucleic acid
hybridized. This information can require a computer for efficient
analysis. The fact of differential fluorescent signal due to
differences in thermal stability of hybrids complicates the
analysis of hybridization results, especially from combinatorial
nucleic acid arrays for de novo sequencing and custom nucleic acid
arrays for specific re-sequencing applications. Modifications in
custom array designs have contributed to simplifying this
problem.
[0006] Further complications can arise and lead to variability in
diagnostic or sequencing results. For example, degradation of
nucleic acid probes, either during the synthesis steps or on
standing can lead to variability in assay results. Accordingly,
there exists a need for additional methods of nucleic acid array
preparation, and the arrays themselves, to provide more robust
tools for the skilled researcher. The present invention provides
such methods and arrays.
SUMMARY OF THE INVENTION
[0007] In one aspect, the present invention provides methods for
preparing nucleic acid arrays on a support. In these methods a
plurality of nucleic acids are synthesized on the support and the
synthesis steps are carried out protected nucleoside
phosphoramidite monomers having less than about 1 mole % of a
phosphoramidite contaminant selected from
(MeO)(NCCH.sub.2CH.sub.2O)PN(iPr).sub.2,
(MeO)P(N(iPr).sub.2).sub.2, (MeO).sub.2PN(iPr).sub.2,
(NCCH.sub.2CH.sub.2O).sub.2PN(iPr).sub.2 or combinations
thereof.
[0008] In one group of embodiments, each nucleic acid occupies a
separate known region of the support, the synthesizing
comprising:
[0009] (a) activating a region of the support;
[0010] (b) attaching a nucleotide to a first region, the nucleotide
having a masked reactive site linked to a protecting group;
[0011] (c) repeating steps (a) and (b) on other regions of the
support whereby each of the other regions has bound thereto another
nucleotide comprising a masked reactive site linked to a protecting
group, wherein the other nucleotide may be the same or different
from that used in step (b);
[0012] (d) removing the protecting group from one of the
nucleotides bound to one of the regions of the support to provide a
region bearing a nucleotide having an unmasked reactive site;
[0013] (e) binding an additional nucleotide to the nucleotide with
an unmasked reactive site;
[0014] (f) repeating steps (d) and (e) on regions of the support
until a desired plurality of nucleic acids is synthesized, each
nucleic acid occupying separate known regions of the support;
[0015] wherein each of steps (a) through (f) are carried out using
nucleoside phosphoramidite monomers having less than about 1 mole %
of a phosphoramidite contaminant, more preferably less than about
0.5 mole % of a phosphoramidite contaminant.
[0016] In another group of embodiments, the preparing comprises the
sequential steps of:
[0017] a) removing a photoremoveable protecting group from at least
a first area of a surface of a substrate, the substrate comprising
immobilized nucleotides on the surface, and the nucleotides capped
with a photoremovable protective group, without removing a
photoremoveable protecting group from at least a second area of the
surface;
[0018] b) simultaneously contacting the first area and the second
area of the surface with a first nucleotide to couple the first
nucleotide to the immobilized nucleotides in the first area, and
not in the second area, the first nucleotide capped with a
photoremovable protective group;
[0019] c) removing a photoremoveable protecting group from at least
a part of the first area of the surface and at least a part of the
second area;
[0020] d) simultaneously contacting the first area and the second
area of the surface with a second nucleotide to couple the second
nucleotide to the immobilized nucleotides in at least a part of the
first area and at least a part of the second area;
[0021] e) performing additional removing and nucleotide contacting
and coupling steps so that a matrix array of at least 100 nucleic
acids having different sequences is formed on the support;
[0022] with the proviso that the phosphoramidite contaminant is
present in an amount of 0.5 mole % or less.
[0023] In another group of embodiments, the nucleoside
phosphoramidite monomers used in the invention have the formula:
1
[0024] wherein B represents adenine, guanine, thymine, cytosine,
uracil or analogs thereof; R is hydrogen, hydroxy, protected
hydroxy, halogen or alkoxy; P is a phosphoramidite group; and PG is
a photoremoveable protected group.
DETAILED DESCRIPTION OF THE INVENTION
[0025] Definitions
[0026] The following definitions are set forth to illustrate and
define the meaning and scope of the various terms used to describe
the invention herein.
[0027] "Nucleic acid library" or "array" is an intentionally
created collection of nucleic acids which run be prepared either
synthetically or biosynthetically and screened for biological
activity in a variety of different formats (e.g., libraries of
soluble molecules; and libraries of oligos tethered to resin beads,
silica chips, or other solid supports). Additionally, the term
"array" is meant to include those libraries of nucleic acids which
can be prepared by spotting nucleic acids of essentially any length
(e.g., from 1 to about 1000 nucleotide monomers in length) onto a
substrate. The term "nucleic acid" as used herein refers to a
polymeric form of nucleotides of any length, either ribonucleotides
or deoxyribonucleotides, that comprise purine and pyrimidine bases,
or other natural, chemically or biochemically modified,
non-natural, or derivatized nucleotide bases. The backbone of the
polynucleotide can comprise sugars and phosphate groups, as may
typically be found in RNA or DNA, or modified or substituted sugar
or phosphate groups. A polynucleotide may comprise modified
nucleotides, such as methylated nucleotides and nucleotide analogs.
The sequence of nucleotides may be interrupted by non-nucleotide
components. Thus the terms nucleoside, nucleotide, deoxynucleoside
and deoxynucleotide generally include analogs such as those
described herein. These analogs are those molecules having some
structural features in common with a naturally occurring nucleoside
or nucleotide such that when incorporated into a nucleic acid or
oligonucleoside sequence, they allow hybridization with a naturally
occurring nucleic acid sequence in solution. Typically, these
analogs are derived from naturally occurring nucleosides and
nucleotides by replacing and/or modifying the base, the ribose or
the phosphodiester moiety. The changes can be tailor made to
stabilize or destabilize hybrid formation or enhance the
specificity of hybridization with a complementary nucleic acid
sequence as desired.
[0028] "Solid support," "support," and "substrate" are used
interchangeably and refer to a material or group of materials
having a rigid or semi-rigid surface or surfaces. In many
embodiments, at least one surface of the solid support will be
substantially flat, although in some embodiments it may be
desirable to physically separate synthesis regions for different
compounds with, for example, wells, raised regions, pins, etched
trenches, or the like. According to other embodiments, the solid
support(s) will take the form of beads, resins, gels, microspheres,
or other geometric configurations.
[0029] "Predefined region" or "preselected region" refers to a
localized area on a solid support which is, was, or is intended to
be used for formation of a selected molecule and is otherwise
referred to herein in the alternative as a "selected" region, a
"known" region, or a "known" location. The predefined or known
region may have any convenient shape, e.g., circular, rectangular,
elliptical, wedge-shaped, etc. For the sake of brevity herein,
"known regions" are sometimes referred to simply as "regions." In
some embodiments, a predefined or known region and, therefore, the
area upon which each distinct compound is synthesized is smaller
than about 1 cm.sup.2 or less than 1 mm.sup.2. Within these
regions, the molecule synthesized therein is preferably synthesized
in a substantially pure form. In additional embodiments, a known
region can be achieved by physically separating the regions (i.e.,
beads, resins, gels, etc.) into wells, trays, etc. Accordingly,
materials (e.g., nucleic acids) can be synthesized or attached to
any particular region by any known methods or means.
[0030] General
[0031] Nucleic acid arrays having single-stranded nucleic acid
probes have become powerful research tools for identifying and
sequencing new genes. Other arrays of unimolecular double-stranded
DNA have been developed which are useful in a variety of screening
assays and diagnostic applications (see, for example, U.S. Pat. No.
5,556,752). Still other arrays have been described in which a
ligand or probe (a peptide, for example), is held in a
conformationally restricted position by two complementary nucleic
acid, at least one of which is attached to a support. Common to
each of these types of arrays is the presence of a support-bound
nucleic acid and the exquisite sensitivity exhibited by the arrays.
Unfortunately, the sensitivity of these arrays can be compromised
if the nucleic acids are not synthesized in sufficient quantity for
assays to provide enough signal relative to background.
[0032] In order to provide the reseacher with arrays of
uncompromising quality and reproducible performance, arrays should
be prepared using high yield reactions and excluding any component
which could negatively impact synthesis yield or the performance of
the array.
[0033] The present invention derives from the discovery that
improved yields and reduced product variablility can be obtained if
nucleic acid arrays are prepared using nucleoside phosphoramidite
monomers that have been purified or prepared in a manner that
exludes interferring impurities such as
(MeO)(NCCH.sub.2CH.sub.2O)PN(iPr).sub.2,
(MeO)P(N(iPr).sub.2).sub.2, (MeO).sub.2PN(iPr).sub.2,
(NCCH.sub.2CH.sub.2O).sub.2PN(iPr).sub.2 or combinations
thereof.
[0034] Embodiments of the Invention
[0035] In view of the above discoveries, the present invention
provides an improved method of preparing a nucleic acid array on a
support. In a general sense, the method comprises synthesizing a
plurality of nucleic acids on a support wherein the synthesis steps
are carried out using protected nucleoside phosphoramidite monomers
having less than about 1 mole % of a phosphoramidite contaminant
selected from the group consisting of
(MeO)(NCCH.sub.2CH.sub.2O)PN(iPr).sub.2,
(MeO)P(N(iPr).sub.2).sub.2, (MeO).sub.2PN(iPr).sub.2, and
(NCCH.sub.2CH.sub.2O).sub.2PN(iPr).sub.2.
[0036] Synthesis of Nucleic Acid Arrays In the present invention,
nucleic acid arrays can be prepared using a variety of synthesis
techniques directed to high-density arrays of nucleic acids on
solid supports. In brief, the methods can include light-directed
methods, flow channel or spotting methods, pin-based methods,
bead-based methods or combinations thereof. For light-directed
methods, see, for example, U.S. Pat. Nos. 5,143,854, 5,424,186 and
5,510,270. For techniques using mechanical methods, see PCT No.
92/10183, U.S. Pat. No. 5,384,261 and PCT/US99/00730. For a
description of bead based techniques, see PCT/US93/04145, and for
pin-based methods, see U.S. Pat. No. 5,288,514. A brief description
of these methods is provided below. The methods of the present
invention are equally amenable to the preparation of unimolecular
double-stranded DNA arrays (see U.S. Pat. No. 5,556,752). In
addition, the nucleic acid arrays prepared in the present methods
will also include those arrays in which individual nucleic acids
are interrupted by non-nucleotide portions (see, for example U.S.
Pat. No. 5,556,752 in which probes such as polypeptides are held in
a conformationally restricted manner by complementary nucleic acid
fragments).
[0037] Various additional techniques for large scale polymer
synthesis are known. Some examples include the U.S. Pat. Nos.
5,143,854, 5,242,979, 5,252,743, 5,324,663, 5,384,261, 5,405,783,
5,412,087, 5,424,186, 5,445,934, 5,451,683, 5,482,867, 5,489,678,
5,491,074, 5,510,270, 5,527,681, 5,550,215, 5,571,639, 5,593,839,
5,599,695, 5,624,711, 5,631,734, 5,677,195, 5,744,101, 5,744,305,
5,753,788, 5,770,456, 5,831,070, and 5,856,011, all of which are
incorporated by reference herein.
[0038] Libraries on a Single Substrate
[0039] Light-Directed Methods
[0040] For those embodiments using a single solid support, the
nucleic acids of the present invention can be formed using
techniques known to those skilled in the art of polymer synthesis
on solid supports. Preferred methods include, for example, "light
directed" methods which are one technique in a family of methods
known as VLSIPS.TM. methods. The light directed methods discussed
in U.S. Pat. No. 5,143,854 involve activating known regions of a
substrate or solid support and then contacting the substrate with a
preselected monomer solution. The known regions can be activated
with a light source, typically shown through a mask (much in the
manner of photolithography techniques used in integrated circuit
fabrication). Other regions of the substrate remain inactive
because they are blocked by the mask from illumination and remain
chemically protected. Thus, a light pattern defines which regions
of the substrate react with a given monomer. By repeatedly
activating different sets of known regions and contacting different
monomer solutions with the substrate, a diverse array of nucleic
acids is produced on the substrate. Of course, other steps such as
washing unreacted monomer solution from the substrate can be used
as necessary.
[0041] The VLSIPS.TM. methods are preferred for the methods
described herein. Additionally, the surface of a solid support,
optionally modified with spacers having photolabile protecting
groups such as NVOC and MeNPOC, is illuminated through a
photolithographic mask, yielding reactive groups (typically
hydroxyl groups) in the illuminated regions. A 3'-O-phosphoramidite
activated deoxynucleoside (protected at the 5'-hydroxyl with a
photolabile protecting group) is then presented to the surface and
chemical coupling occurs at sites that were exposed to light.
Following capping, and oxidation, the substrate is rinsed and the
surface illuminated through a second mask, to expose additional
hydroxyl groups for coupling. A second 5'-protected,
3'-O-phosphoramidite activated deoxynucleoside is presented to the
surface. The selective photodeprotection and coupling cycles are
repeated until the desired set of nucleic acids is produced.
Alternatively, an oligomer of from, for example, 4 to 30
nucleotides can be added to each of the preselected regions rather
than synthesize each member in a monomer by monomer approach.
Methods for light-directed synthesis of DNA arrays on glass
substrates are also described in McGall et al., J. Am. Chem. Soc.,
119:5081-5090 (1997).
[0042] For the above light-directed methods wherein photolabile
protecting groups and photolithography are used to create spatially
addressable parallel chemical synthesis of a nucleic acid array
(see also U.S. Pat. No. 5,527,681), computer tools may be used to
assist in forming the arrays. For example, a computer system may be
used to select nucleic acid or other polymer probes on the
substrate, and design the layout of the array as described in, for
example, U.S. Pat. No. 5,571,639.
[0043] Flow Channel or Spotting Methods
[0044] Additional methods applicable to library synthesis on a
single substrate are described in U.S. Pat. No. 5,384,261 and in
PCT/US99/00730. In the methods disclosed in this patent and PCT
publication, reagents are delivered to the substrate by either (1)
flowing within a channel defined on known regions or (2) "spotting"
on known regions. However, other approaches, as well as
combinations of spotting and flowing, may be employed. In each
instance, certain activated regions of the substrate are
mechanically separated from other regions when the monomer
solutions are delivered to the various reaction sites.
[0045] A typical "flow channel" method applied to the compounds and
libraries of the present invention can generally be described as
follows. Diverse nucleic acid sequences are synthesized at selected
regions of a substrate or solid support by forming flow channels on
a surface of the substrate through which appropriate reagents flow
or in which appropriate reagents are placed. For example, assume a
monomer "A" is to be bound to the substrate in a first group of
selected regions. If necessary, all or part of the surface of the
substrate in all or a part of the selected regions is activated for
binding by, for example, flowing appropriate reagents through all
or some of the channels, or by washing the entire substrate with
appropriate reagents. After placement of a channel block on the
surface of the substrate, a reagent having the monomer A flows
through or is placed in all or some of the channel(s). The channels
provide fluid contact to the first selected regions, thereby
binding the monomer A on the substrate directly or indirectly (via
a spacer) in the first selected regions.
[0046] Thereafter, a monomer B is coupled to second selected
regions, some of which maybe included among the first selected
regions. The second selected regions will be in fluid contact with
a second flow channel(s) through translation, rotation, or
replacement of the channel block on the surface of the substrate;
through opening or closing a selected valve; or through deposition
of a layer of chemical or photoresist. If necessary, a step is
performed for activating at least the second regions. Thereafter,
the monomer B is flowed through or placed in the second flow
channel(s), binding monomer B at the second selected locations. In
this particular example, the resulting sequences bound to the
substrate at this stage of processing will be, for example, A, B,
and AB. The process is repeated to form a vast array of sequences
of desired length at known locations on the substrate.
[0047] After the substrate is activated, monomer A can be flowed
through some of the channels, monomer B can be flowed through other
channels, a monomer C can be flowed through still other channels,
etc. In this manner, many or all of the reaction regions are
reacted with a monomer before the channel block must be moved or
the substrate must be washed and/or reactivated. By making use of
many or all of the available reaction regions simultaneously, the
number of washing and activation steps can be minimized.
[0048] One of skill in the art will recognize that there are
alternative methods of forming channels or otherwise protecting a
portion of the surface of the substrate. For example, according to
some embodiments, a protective coating such as a hydrophilic or
hydrophobic coating (depending upon the nature of the solvent) is
utilized over portions of the substrate to be protected, sometimes
in combination with materials that facilitate wetting by the
reactant solution in other regions. In this manner, the flowing
solutions are further prevented from passing outside of their
designated flow paths.
[0049] The "spotting" methods of preparing nucleic acid libraries
can be implemented in much the same manner as the flow channel
methods. For example, a monomer A can be delivered to and coupled
with a first group of reaction regions which have been
appropriately activated. Thereafter, a monomer B can be delivered
to and reacted with a second group of activated reaction regions.
Unlike the flow channel embodiments described above, reactants are
delivered by directly depositing (rather than flowing) relatively
small quantities of them in selected regions. In some steps, of
course, the entire substrate surface can be sprayed or otherwise
coated with a solution. In preferred embodiments, a dispenser moves
from region to region, depositing only as much monomer as necessary
at each stop. Typical dispensers include a micropipette to deliver
the monomer solution to the substrate and a robotic system to
control the position of the micropipette with respect to the
substrate, or an ink jet printer. In other embodiments, the
dispenser includes a series of tubes, a manifold, an array of
pipettes, or the like so that various reagents can be delivered to
the reaction regions simultaneously. Still other spotting methods
are described in PCT/US99/00730.
[0050] Pin-Based Methods
[0051] Another method which is useful for the preparation of
nucleic acid arrays and libraries involves "pin based synthesis."
This method is described in detail in U.S. Pat. No. 5,288,514. The
method utilizes a substrate having a plurality of pins or other
extensions. The pins are each inserted simultaneously into
individual reagent containers in a tray. In a common embodiment, an
array of 96 pins/containers is utilized.
[0052] Each tray is filled with a particular reagent for coupling
in a particular chemical reaction on an individual pin.
Accordingly, the trays will often contain different reagents. Since
the chemistry disclosed herein has been established such that a
relatively similar set of reaction conditions may be utilized to
perform each of the reactions, it becomes possible to conduct
multiple chemical coupling steps simultaneously. In the first step
of the process the invention provides for the use of substrate(s)
on which the chemical coupling steps are conducted. The substrate
is optionally provided with a spacer having active sites. In the
particular case of nucleic acids, for example, the spacer may be
selected from a wide variety of molecules which can be used in
organic environments associated with synthesis as well as aqueous
environments associated with binding studies. Examples of suitable
spacers are polyethyleneglycols, dicarboxylic acids, polyamines and
alkylenes, substituted with, for example, methoxy and ethoxy
groups. Additionally, the spacers will have an active site on the
distal end. The active sites are optionally protected initially by
protecting groups. Among a wide variety of protecting groups which
are useful are FMOC, BOC, t-butyl esters, t-butyl ethers, and the
like. Various exemplary protecting groups are described in, for
example, Atherton et al., SOLID PHASE PEPTIDE SYNTHESIS, IRL Press
(1989). In some embodiments, the spacer may provide for a cleavable
function by way of, for example, exposure to acid or base.
[0053] Libraries on Multiple Substrates
[0054] Bead Based Methods
[0055] Yet another method which is useful for synthesis of nucleic
acid arrays involves "bead based synthesis." A general approach for
bead based synthesis is described in PCT/US93/04145 (filed Apr. 28,
1993).
[0056] For the synthesis of nucleic acids on beads, a large
plurality of beads are suspended in a suitable carrier (such as
water) in a container. The beads are provided with optional spacer
molecules having an active site. The active site is protected by an
optional protecting group.
[0057] In a first step of the synthesis, the beads are divided for
coupling into a plurality of containers. For the purposes of this
brief description, the number of containers will be limited to
three, and the monomers denoted as A, B, C, D, E, and F. The
protecting groups are then removed and a first portion of the
molecule to be synthesized is added to each of the three containers
(i.e., A is added to container 1, B is added to container 2 and C
is added to container 3).
[0058] Thereafter, the various beads are appropriately washed of
excess reagents, and remixed in one container. Again, it will be
recognized that by virtue of the large number of beads utilized at
the outset, there will similarly be a large number of beads
randomly dispersed in the container, each having a particular first
portion of the monomer to be synthesized on a surface thereof.
[0059] Thereafter, the various beads are again divided for coupling
in another group of three containers. The beads in the first
container are deprotected and exposed to a second monomer (D),
while the beads in the second and third containers are coupled to
molecule portions E and F respectively. Accordingly, molecules AD,
BD, and CD will be present in the first container, while AE, BE,
and CE will be present in the second container, and molecules AF,
BF, and CF will be present in the third container. Each bead,
however, will have only a single type of molecule on its surface.
Thus, all of the possible molecules formed from the first portions
A, B, C, and the second portions D, E, and F have been formed.
[0060] The beads are then recombined into one container and
additional steps such as are conducted to complete the synthesis of
the polymer molecules. In a preferred embodiment, the beads are
tagged with an identifying tag which is unique to the particular
nucleic acid or probe which is present on each bead. A complete
description of identifier tags for use in synthetic libraries is
provided in co-pending application Ser. No. 08/146,886 (filed Nov.
2, 1993).
[0061] Solid Supports
[0062] Solid supports used in the present invention include any of
a variety of fixed organizational support matrices. In some
embodiments, the support is substantially planar. In some
embodiments, the support may be physically separated into regions,
for example, with trenches, grooves, wells and the like. Examples
of supports include slides, beads and solid chips. Additionally,
the solid supports may be, for example, biological, nonbiological,
organic, inorganic, or a combination thereof, and may be in forms
including particles, strands, gels, sheets, tubing, spheres,
containers, capillaries, pads, slices, films, plates, and slides
depending upon the intended use.
[0063] Supports having a surface to which arrays of nucleic acids
are attached are also referred to herein as "biological chips." The
support is preferably, silica or glass, and can have the thickness
of a microscope slide or glass cover slip. Supports that are
transparent to light are useful when the assay involves optical
detection, as described, e.g., in U.S. Pat. No. 5,545,531. Other
useful supports include Langmuir Blodgett film, germanium,
(poly)tetrafluorethylene, polystyrene, (poly)vinylidenedifluoride,
polycarbonate, gallium arsenide, gallium phosphide, silicon oxide,
silicon nitride, and combinations thereof. In one embodiment, the
support is a flat glass or single crystal silica surface with
relief features less than about 10 Angstroms.
[0064] The surfaces on the solid supports will usually, but not
always, be composed of the same material as the substrate. Thus,
the surface may comprise any number of materials, including
polymers, plastics, resins, polysaccharides, silica or silica based
materials, carbon, metals, inorganic glasses, membranes, or any of
the above-listed substrate materials. Preferably, the surface will
contain reactive groups, such as carboxyl, amino, and hydroxyl. In
one embodiment, the surface is optically transparent and will have
surface Si--OH functionalities such as are found on silica
surfaces. In other embodiments, the surface will be coated with
functionalized silicon compounds (see, for example, U.S. Pat. No.
5,919,523).
[0065] Surface Density
[0066] The nucleic acid arrays described herein can have any number
of nucleic acid sequences selected for different applications.
Typically, there may be, for example, about 100 or more, or in some
embodiments, more than 10.sup.5 or 10.sup.8. In one embodiment, the
surface comprises at least 100 probe nucleic acids each preferably
having a different sequence, each probe contained in an area of
less than about 0.1 cm.sup.2, or, for example, between about
1/mm.sup.2 and 10,000/mm.sup.2, and each probe nucleic acid having
a defined sequence and location on the surface. In one embodiment,
at least 1,000 different nucleic acids are provided on the surface,
wherein each nucleic acid is contained within an area less than
about 10.sup.-3 cm.sup.2, as described, for example, in U.S. Pat.
No. 5,510,270.
[0067] Arrays of nucleic acids for use in gene expression
monitoring are described in PCT WO 97/10365, the disclosure of
which is incorporated herein. In one embodiment, arrays of nucleic
acid probes are immobilized on a surface, wherein the array
comprises more than 100 different nucleic acids and wherein each
different nucleic acid is localized in a predetermined area of the
surface, and the density of the different nucleic acids is greater
than about 60 different nucleic acids per 1 cm.sup.2.
[0068] Arrays of nucleic acids immobilized on a surface which may
be used also are described in detail in U.S. Pat. No. 5,744,305,
the disclosure of which is incorporated herein. As disclosed
therein, on a substrate, nucleic acids with different sequences are
immobilized each in a known area on a surface. For example, 10, 50,
60, 100, 10.sup.3, 10.sup.4, 10.sup.5, 10.sup.6, 10.sup.7, or
10.sup.8 different monomer sequences may be provided on the
substrate. The nucleic acids of a particular sequence are provided
within a known region of a substrate, having a surface area, for
example, of about 1 cm.sup.2 to 10.sup.-10 cm.sup.2. In some
embodiments, the regions have areas of less than about 10.sup.-1,
10.sup.-2, 10.sup.-3, 10.sup.-4, 10.sup.-5, 10.sup.-6, 10.sup.-7,
10.sup.-8, 10.sup.-9, or 10.sup.-10 cm.sup.2. For example, in one
embodiment, there is provided a planar, non-porous support having
at least a first surface, and a plurality of different nucleic
acids attached to the first surface at a density exceeding about
400 different nucleic acids/cm.sup.2, wherein each of the different
nucleic acids is attached to the surface of the solid support in a
different known region, has a different determinable sequence, and
is, for example, at least 4 nucleotides in length. The nucleic
acids may be, for example, about 4 to 20 nucleotides in length. The
number of different nucleic acids may be, for example, 1000 or
more. In the embodiment where polynucleotides of a known chemical
sequence are synthesized at known locations on a substrate, and
binding of a complementary nucleotide is detected, and wherein a
fluorescent label is detected, detection may be implemented by
directing light to relatively small and precisely known locations
on the substrate. For example, the substrate is placed in a
microscope detection apparatus for identification of locations
where binding takes place. The microscope detection apparatus
includes a monochromatic or polychromatic light source for
directing light at the substrate, means for detecting fluoresced
light from the substrate, and means for determining a location of
the fluoresced light. The means for detecting light fluoresced on
the substrate may in some embodiments include a photon counter. The
means for determining a location of the fluoresced light may
include an x/y translation table for the substrate. Translation of
the substrate and data collection are recorded and managed by an
appropriately programmed digital computer, as described in U.S.
Pat. No. 5,510,270.
[0069] Applications Using Nucleic Acid Arrays
[0070] The methods and compositions described herein may be used in
a range of applications including biomedical and genetic research
as well as clinical diagnostics. Arrays of polymers such as nucleic
acids may be screened for specific binding to a target, such as a
complementary nucleotide, for example, in screening studies for
determination of binding affinity and in diagnostic assays. In one
embodiment, sequencing of polynucleotides can be conducted, as
disclosed in U.S. Pat. No. 5,547,839. The nucleic acid arrays may
be used in many other applications including detection of genetic
diseases such as cystic fibrosis, diabetes, and acquired diseases
such as cancer, as disclosed in U.S. patent application Ser. No.
08/143,312. Genetic mutations may be detected by sequencing by
hydridization. In one embodiment, genetic markers may be sequenced
and mapped using Type-IIs restriction endonucleases as disclosed in
U.S. Pat. No. 5,710,000.
[0071] Other applications include chip based genotyping, species
identification and phenotypic characterization, as described in
U.S. patent application Ser. No. 08/797,812, filed Feb. 7, 1997,
and U.S. application Ser. No. 08/629,031, filed Apr. 8, 1996. Still
other applications are described in U.S. Pat. No. 5,800,992.
[0072] Gene expression may be monitored by hybridization of large
numbers of mRNAs in parallel using high density arrays of nucleic
acids in cells, such as in microorganisms such as yeast, as
described in Lockhart et al., Nature Biotechnology, 14:1675-1680
(1996). Bacterial transcript imaging by hybridization of total RNA
to nucleic acid arrays may be conducted as described in Saizieu et
al., Nature Biotechnology, 16:45-48 (1998). Accessing genetic
information using high density DNA arrays is further described in
Chee, Science 274:610-614 (1996).
[0073] Still other methods for screening target molecules for
specific binding to arrays of polymers, such as nucleic acids,
immobilized on a solid substrate, are disclosed, for example, in
U.S. Pat. No. 5,510,270. The fabrication of arrays of polymers,
such as nucleic acids, on a solid substrate, and methods of use of
the arrays in different assays, are also described in: U.S. Pat.
Nos. 5,677,195, 5,624,711, 5,599,695, 5,445,934, 5,451,683,
5,424,186, 5,412,087, 5,405,783, 5,384,261, 5,252,743 and
5,143,854; PCT WO 92/10092; and U.S. application Ser. No.
08/388,321, filed Feb. 14, 1995.
[0074] Devices for concurrently processing multiple biological chip
assays are useful for each of the applications described above
(see, for example, U.S. Pat. No. 5,545,531). Methods and systems
for detecting a labeled marker on a sample on a solid support,
wherein the labeled material emits radiation at a wavelength that
is different from the excitation wavelength, which radiation is
collected by collection optics and imaged onto a detector which
generates an image of the sample, are disclosed in U.S. Pat. No.
5,578,832. These methods permit a highly sensitive and resolved
image to be obtained at high speed. Methods and apparatus for
detection of fluorescently labeled materials are further described
in U.S. Pat. Nos. 5,631,734 and 5,324,633.
[0075] Preferred Embodiments
[0076] In view of the technologies provided above, the present
invention provides in one preferred embodiment, a method of
preparing a nucleic acid array on a support, wherein each nucleic
acid occupies a separate known region of the support and the
nucleic acids are synthesized using the steps:
[0077] (a) activating a region of the support;
[0078] (b) attaching a nucleotide to a first region, the nucleotide
having a masked reactive site linked to a protecting group;
[0079] (c) repeating steps (a) and (b) on other regions of the
support whereby each of the other regions has bound thereto another
nucleotide comprising a masked reactive site link to a protecting
group, wherein the another nucleotide may be the same or different
from that used in step (b);
[0080] (d) removing the protecting group from one of the
nucleotides bound to one of the regions of the support to provide a
region bearing a nucleotide having an unmasked reactive site;
[0081] (e) binding an additional nucleotide to the nucleotide with
an unmasked reactive site;
[0082] (f) repeating steps (d) and (e) on regions of the support
until a desired plurality of nucleic acids is synthesized, each
nucleic acid occupying separate known regions of the support;
[0083] wherein the nucleotides used in steps (b) through (f) are
protected nucleoside phosphoramidite monomers having less than
about 1 mole % of a phosphoramidite contaminant selected from
(Me0)(NCCH.sub.2CH.sub.2O)PN(iP- r).sub.2,
(MeO)P((iPr).sub.2).sub.2, (MeO).sub.2PN(iPr).sub.2,
(NCCH.sub.2CH.sub.2O).sub.2PN(iPr).sub.2 or combinations
thereof.
[0084] Preferably, the "activating" of step (a) is carried out
using a channel block or photolithography technique, more
preferably a photolithography technique. The "attaching" of step
(b) is typically carried out using chemical means to provide a
covalent bond between the nucleotide and a surface functional group
present in the first region. In some embodiments, the surface
functional group will be a group present on a nucleotide or nucleic
acid that is already attached to the solid support. For example,
nucleic acid arrays can be prepared using a solid support having a
surface coated with poly-A nucleic acids to provide suitable
spacing between the surface of the support and the nucleic acids
that will be used in subsequent hybridization assays. Accordingly,
the "attaching" can be, for example, by formation of a covalent
bond between surface Si--OH groups and a group present on the first
nucleotide of a nascent nucleic acid chain, or by formation of a
covalent bond between groups present in a support-bound nucleic
acid and a group present on the first nucleotide of a nascent
nucleic acid. Typically, the groups present on nucleic acids which
are used in covalent bond formation are the 3'- or 5-hydroxyl
groups in the sugar portion or the molecule, or phosphate groups
attached thereto. The nucleotides used in this and other aspects of
the present invention will typically be the naturally-occuring
nucleotides, derived from, for example, adenosine, guanosine,
uridine, cytidine,and thymidine. In certain embodiments, however,
nucleotide analogs or derivatives will be used (e.g., those
nucleosides or nucleotides having protecting groups on either the
base portion or sugar portion of the molecule, or having attached
or incorporated labels, or isosteric replacements which result in
monomers that behave in either a synthetic or physiological
environment in a manner similar to the parent monomer). The
nucleotides will typically have a protecting group which is linked
to, and masks, a reactive group on the nucleotide. A variety of
protecting groups are useful in the invention and can be selected
depending on the synthesis techniques employed. For example,
channel block methods can use acid- or base-cleavable protecting
groups to mask a hydroxyl group in a nucleotide. After the
nucleotide is attached to the support or growing nucleic acid, the
protecting group can be removed by flowing an acid or base solution
through an appropriate channel on the support.
[0085] Similarly, photolithography techniques can use
photoremoveable protecting groups. Some classes of photoremovable
protecting groups include 6-nitroveratryl (NV), 6-nitropiperonyl
(NP), methyl-6-nitroveratryl (MeNV), methyl-6-nitropiperonyl
(MeNP), and 1-pyrenylmethyl (PyR), which are used for protecting
the carboxyl terminus of an amino acid, or the hydroxyl group of a
nucleotide, for example. 6-nitroveratryloxycarbonyl (NVOC),
6-nitropiperonyloxycarbonyl (NPOC),
methyl-6-nitroveratryloxycarbonyl (MeNVOC),
methyl-6-nitropiperonyloxycarbonyl (MeNPOC),
1-pyrenylmethyloxycarbonyl (PyROC), which are used to protect the
amino terminus of an amino acid are also preferred. Clearly, many
photosensitive protecting groups are suitable for use in the
present invention (see, U.S. Pat. No. 5,489,678 and PCT WO
94/10128).
[0086] In addition, novel photoremovable protecting groups such as
5'-O-pyrenylmethyloxy carbonyl (PYMOC) and
methylnitropiperonyloxycarbony- l (MeNPOC) have been described in
U.S. patent application Ser. No. 08/630,148, filed Apr. 10, 1996,
the contents of which are hereby incorporated by reference.
[0087] In addition to the above-described protecting groups, the
present invention employs protecting groups, such as the
5'-X-2'-deoxythymidine 2-cyanoethyl
3'-N,N-diisopropylphosphoramidites in various solvents. In these
protecting groups, X may represent the following photolabile
groups: ((.alpha.-methyl-2-nitropiperonyl)-oxy)carbonyl (MeNPOC),
((Phenacyl)-oxy)carbonyl (PAOC), O-(9-phenylxanthen-9-yl) (PIXYL),
and ((2-methylene-9,10-anthraquinone)-oxy)carbonyl (MAQOC).
[0088] Various methods for generating protected monomers have been
described by the U.S. Pat. No. 5,744,305, which is incorporated by
reference. Detailed methods for using photoremovable protecting
groups are described in the U.S. Pat. No. 5,424,186, which is also
hereby incorporated by reference.
[0089] The removal rate of the protecting groups depends on the
wavelength and intensity of the incident radiation, as well as the
physical and chemical properties of the protecting group itself.
Preferred protecting groups are removed at a faster rate and with a
lower intensity of radiation. For example, at a given set of
conditions, MeNVOC and MeNPOC are photolytically removed faster
than their unsubstituted parent compounds, NVOC and NPOC,
respectively.
[0090] In addition to the above-described references,
photocleavable protecting groups and methods of using such
photocleavable protecting groups for polymer synthesis have been
described in U.S. patent application Ser. Nos. 08/630,148 (filed
Apr. 10, 1996) and 08/812,005 (filed Mar. 5, 1997) which are
incorporated by reference herein.
[0091] Step (c) provides that steps (a) and (b) can be repeated to
attach nucleotides to other regions of the solid support.
[0092] One of skill in the art will appreciate that steps (a) and
(b) can be repeated a number of times to produce a solid support
having a layer of attached nucleotides. Preferably, each attached
nucleotide is in a known position.
[0093] In subsequent steps (d), (e) and (f), the protecting group
is removed from one of the nucleotides to reveal a reactive site on
the nucleotide. Thereafter, an additional nucleotide (optionally
having a masked reactive site attached to a protecting group) is
attached to the support-bound nucleotide. As above, these steps can
be repeated to selectively attach an additional nucleotide to any
of the support-bound nucleotides. Still further, the steps of
deprotecting and attaching an additional nucleotide can be carried
out on the newly added nucleotides to continue the synthesis of the
nascent nucleic acid.
[0094] As noted above, the above steps are preferably carried out
using protected nucleoside phosphoramidite monomers having less
than about 1 mole % of a phosphoramidite contaminant selected from
(MeO)(NCCH.sub.2CH.sub.2O)PN(iPr).sub.2,
(MeO)P(N(iPr).sub.2).sub.2, (MeO).sub.2PN(iPr).sub.2,
(NCCH.sub.2CH.sub.2O).sub.2PN(iPr).sub.2 or combinations thereof.
More preferably, the contaminant is present with the monomer or
monomer solution in an amount of about 0.5 mole % or less, most
preferably in an amount of about 0.2 mole % or less.
[0095] In a further preferred embodiment, the preparing
comprises:
[0096] a) removing a photoremoveable protecting group from at least
a first area of a surface of a substrate, the substrate comprising
immobilized nucleotides on the surface, and the nucleotides capped
with a photoremovable protective group, without removing a
photoremoveable protecting group from at least a second area of the
surface;
[0097] b) simultaneously contacting the first area and the second
area of the surface with a first nucleotide to couple the first
nucleotide to the immobilized nucleotides in the first area, and
not in the second area, the first nucleotide capped with a
photoremovable protective group;
[0098] c) removing a photoremoveable protecting group from at least
a part of the first area of the surface and at least a part of the
second area;
[0099] d) simultaneously contacting the first area and the second
area of the surface with a second nucleotide to couple the second
nucleotide to the immobilized nucleotides in at least a part of the
first area and at least a part of the second area;
[0100] e) performing additional removing and nucleotide contacting
and coupling steps so that a matrix array of at least 100 nucleic
acids having different sequences is formed on the support;
[0101] with the proviso that the phosphoramidite contaminant is
present in an amount of 0.5 mole % or less.
[0102] In this embodiment of the invention, the steps of removing
photoremoveable protecting groups, coupling nucleotides to specific
areas, removing protecting groups from the coupled nucleotides, and
coupling additional nucleotides can all be carried out as described
in, for example, U.S. Pat. No. 5,510,270, with the added feature
that the coupling steps are performed using protected nucleoside
phosphoramidite monomers that are contaminated by less than about 1
mole % of a phosphoramidite contaminant such as
(MeO)(NCCH.sub.2CH.sub.2O)PN(iPr).sub- .2,
(MeO)P(N(iPr).sub.2).sub.2, (MeO).sub.2PN(iPr).sub.2,
(NCCH.sub.2CH.sub.2O).sub.2PN(iPr).sub.2 or combinations
thereof.
[0103] In still further preferred embodiments, the nucleoside
phosphoramidite monomers used in the methods described above have
the formula: 2
[0104] wherein B represents adenine, guanine, thymine, cytosine,
uracil or analogs thereof; R is hydrogen, hydroxy, protected
hydroxy, halogen or alkoxy; P is a phosphoramidite group; and PG is
a photoremoveable protected group.
[0105] In the group of embodiments using monomers of formula (I), B
is preferably adenine, guanine, thymine, cytosine or uracil. More
preferably, B is adenine, guanine, thymine, or cytosine, and R is
hydrogen. Still more preferably, the array prepared using the
monomers above comprises at least 10 different nucleid acids, more
preferably at least 100 different nucleic acids, still more
preferably at least 1000 different nucleic acids. Most preferably,
the array comprises at least 10,000 to 100,000 or more different
nucleic acids. Additionally, each different nucleic acid is in a
region having an area of less than about 1 cm.sup.2, more
preferably less than about 1 mm.sup.2.
[0106] In still other preferred embodiments, B is adenine, guanine,
thymine, or cytosine; R is hydrogen; and the phosphoramidite
contaminant is present in an amount of less than 0.2 mole %. More
preferably, B is adenine, guanine, thymine, or cytosine; R is
hydrogen; PG is MeNPOC and the phosphoramidite contaminant is
present in an amount of less than 0.2 mole %. Still more
preferably, B is adenine, guanine, thymine, or cytosine; R is
hydrogen; PG is MeNPOC, P is --P(OCH.sub.2CH.sub.2CN)N(iPr- ).sub.2
and the phosphoramidite contaminant is present in an amount of less
than 0.2 mole %.
[0107] One of skill in the art will appreciate that the present
invention can be readily modified to use protected nucleoside
phospohoramidite monomers wherein the protecting group on the 5'
hydroxy is acid or base removeable. Such modifications will render
the invention applicable to other synthesis methodologies such as
flow channel and spotting methods described in more detail above.
Regardless of the array synthesis methods, removal of competing
phosphoramidite impurities can dramatically increase the yield of
nucleic acid synthesis on the substrate.
EXAMPLES
[0108] In each of the examples below, the nucleic acid probe arrays
were prepared using photolithography and a silica wafer as the
solid substrate. Preparation is typically on a 5 inch by 5 inch
wafer which can be cut into 49 replicates of a probe array having
about 400,000 distinct probe sequences, or 400 replicates of a
probe array having about 50,000 distinct probe sequences. The
density of the nucleic acid probes is about 1-10 picomoles per
cm.sup.2.
Example 1
[0109] This example illustrates the analysis, detection of impurity
and purification of a protected nucleoside phosphoramidite.
[0110] One lot of MeNPOC-dC.sup.ibu-CEP
(MeNPOC-N.sup.4-isobutyryl-2'deoxy- cytidine-CEP, wherein the
MeNPOC group is attached to the 5'-OH and the cyanoethyl
phosphoramidite (CEP) is attached to the 3'-OH) was found to be in
conformance with analytical specifications, but failed in coupling
efficiency tests with a 6-mer synthesis yield of only 14% of
control. In these efficiency tests, the synthesis involved
preparation of C6 nucleic acids on a silica support using
photolithographic techniques. Quantitation of the hexamer produced
indicated a coupling efficiency (% yield for the six steps) which
was typically about 16-32% when purified phosphoramidite solutions
were used, versus a coupling efficiency of about 2.8 to 3.6% when
the phosphoramidite solution contained an impurity (identified
below).
[0111] The nucleic acids synthesized were removed from the support
and HPLC analysis of the synthesis products showed truncated
nucleic acids with abnormal retention times. A mixing experiment
was carried out which gave a result consistent with the presence of
an impurity in the lot of MeNPOC-dC.sup.ibu-CEP. The impurity was
not detectable by .sup.31P-NMR or by HPLC. Close examination of the
.sup.1H-NMR revealed a signal (relative to tetramethylsilane) at
3.42 ppm (doublet, J=3.8 Hz). The signal corresponded to a small
amount (.about.3-5 mole %) of
(MeO)(NCCH.sub.2CH.sub.2O)PN(iPr).sub.2, or "methyl-CEP" as a
contaminant.
[0112] Methyl-CEP was found to be an aggressive capping agent,
competing for and blocking surface sites at a much faster rate than
the C-phosphoramidite itself.
[0113] Methyl-CEP can be removed from lots of MeNPOC-dC.sup.ibu-CEP
by silica gel chromatography.
Example 2
[0114] This example illustrates the removal of Methyl-CEP from a
lot of MeNPOC-dC.sub.ibu-CEP.
[0115] Flash Column
[0116] Silica gel 60 was pre-wetted with 1% triethylaminelethyl
acetate for at least one hour prior to chromatography. A flash
column was packed with 50.times.30 mm (length.times.width) of the
wet silica and the column was washed with 10 column volumes of
ethyl acetate. The column was charged with 0.35 g of
MeNPOC-dC.sup.ibu-CEP dissolved in a minimum amount of
dichloromethane containing 0.05% triethylamine.
[0117] Chromatography
[0118] The amidite (MeNPOC-dC.sup.ibu-CEP) was eluted with ethyl
acetate and collected in 5 mL fractions. The leading and tailing
fractions were discarded, and the remaining fractions were pooled.
Solvent was evaporated and the residue was co-evaporated with
anhydrous acetonitrile (3.times.), then dried under high vacuum for
1 hour to afford 260 mg (74% recovery) of MeNPOC-dC.sup.ibu-CEP as
a pale yellow glass. The amidite was stored in the dark at
-20.degree. C.
[0119] Analysis
[0120] The purified material was analyzed against crude material by
TLC on silica gel 60 f254 plates pre-treated with 1% triethylamine
in ethyl acetate. The plate was eluted with 7:3 ethyl
acetate:hexanes containing 1% triethylamine. Additional analysis
using .sup.1H-NMR and .sup.31P-NMR (sample in CDCl.sub.3) showed no
detectable peaks corresponding to the impurity.
[0121] It is understood that the examples and embodiments described
herein are for illustrative purposes only and that various
modifications or changes in light thereof will be suggested to
persons skilled in the art and are to be included within the spirit
and purview of this application and scope of the appended claims.
All publications, patents, and patent applications cited herein are
hereby incorporated by reference in their entirety for all
purposes.
[0122] While this invention has been particularly shown and
described with references to preferred embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
scope of the invention encompassed by the appended claims.
* * * * *